Methods for treating a corneal disorder and promoting skin wound healing

ABSTRACT

A method for treating a corneal disorder or for promoting skin wound healing comprising administering to a patient in need thereof a composition containing pharmaceutically effective amounts of (i) a first peptide comprising an amino acid sequence containing the following consecutive amino acids: Ser-Ser-Ser-Arg (SEQ ID NO: 1), or an analog thereof or a pharmaceutically acceptable salt thereof and (ii) a second peptide comprising an amino acid sequence containing the following consecutive amino acids with a terminal —NH 2  group: Phe-Gly-Leu-Met-NH 2  (SEQ ID NO: 2), or an analog thereof or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of application Ser. No.10/497,628 filed Jun. 3, 2004 (U.S. Pat. No. 7,232,881), which is theUnited States national phase application of International ApplicationPCT/JP02/12632 filed Dec. 3, 2002.

TECHNICAL FIELD

The present invention relates to a novel peptide containing the aminoacid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1), apharmaceutical composition containing the peptide as the activeingredient, and a therapeutic agent of corneal disorders or an agentpromoting skin wound healing which comprises a peptide containing theamino acid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) and apeptide containing the amino acid sequence represented byPhe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2)as the active ingredients.

BACKGROUND OF THE INVENTION

Cornea is a transparent, non-vascular tissue having a diameter of about1 cm and a thickness of about 1 mm. The transparency of cornea affectsthe visionary function. Various physiological and biochemical phenomenain cornea work functionally, mainly for the purpose of maintaining thetransparency of cornea.

Corneal epithelial defects caused by various diseases such as cornealulcer, corneal erosion, keratitis and dry eye is spontaneously repairedunless mixed infection is associated. When the repair is delayed or notcompleted or the epithelial defect is prolonged with some reason,however, not only the normal construction of corneal epithelium is badlyaffected but also even the constructions and functions of the cornealstroma and endothelium are damaged. The principle of the therapeuticmethods according to the conventional art is passive. That is, thetherapeutic methods include protecting the corneal surface from externalstimulation to intend spontaneous extension of corneal epithelium forre-surfacing the defected area. Following the development of cellbiology in recent years, factors involved in proliferation, migration,adhesion, extension and the like have been elucidated. It was reportedthat compounds promoting the extension of corneal epithelium playimportant roles in repairing corneal epithelial defects (JapaneseJournal of Clinical Ophthalmology, 46, 738-743 (1992); Japanese Journalof Ophthalmologic Surgery, 5, 719-727 (1992)).

Meanwhile, insulin-like growth factor is one of growth factorsregulating growth of normal human cells like epidermal growth factors,fibroblast growth factors, platelet-derived growth factors andtransforming growth factors and includes insulin-like growth factor-I(referred to as “IGF-I” hereinafter) and insulin-like growth factor-II(referred to as “IGF-II” hereinafter). Recently, for example, it wasreported that IGF-I stimulates the proliferation of thyroid cells (J.Biol. Chem., 264, 18485-18488 (1989)) and that IGF-II regulates themuscle growth and differentiation (Hum. Mol. Genet., 3, 1117-1121(1994)). In the field of ophthalmology, it was disclosed that IGF-I,IGF-II and their functional derivatives promote the survival of retinalneurons (the publication of Japanese Patent Publication (Tokuhyo)7-500839); that IGF-II is widely effective for the treatment of alltypes of wounds mainly including lesions made during keratoplasty (thepublication of JP-A-63-233925); and that a solution containing thegrowth factors can be used to keep eye tissues such as cornea to be usedfor keratoplasty at their fresh state even in a circumstance at low atemperature (the publications of JP-A-5-25001 and JP-A-6-48901).Further, another disclosure is made about a gel composition containing agrowth factor that the gel composition is generally effective for woundhealing of for example the anterior segment of the eye (the publicationof JP-A-2-112).

On the other hand, substance P is a polypeptide consisting of 11 aminoacids and has actions such as vasodilatation, the smooth musclecontraction, the promotion of salivary gland secretion, and diuresis. Inthe field of ophthalmology, it is disclosed that substance P can improveabnormal secretion of the goblet cells of conjunctiva (the publicationof International Publication WO95/13087), while the kinetics ofsubstance P in the case of inflammation such as keratitis is alsoreported (Nippon Ganka Gakkai Zasshi, 91, 982-987 (1987); Nippon GankaGakkai Zasshi, 92, 448-452 (1988); and the like). As described above,various studies have been done. Additionally, the publication ofJP-A-10-17489 describes that the tetrapeptide Phe-Gly-Leu-Met-NH₂ (SEQID NO: 2) (referred to as “FGLM” hereinafter) on the C terminal ofsubstance P when used in combination with IGF-I can promote woundhealing of corneal epithelium and that the FGLM (SEQ ID NO: 2) is theminimum unit among partial peptides with such action of substance P.However, it has never been identified yet which amino acid sequence sitein IGF-1 is responsible for the expression of the effect while IGF-1 isa polypeptide consisting of amino acids as many as 70.

Skin wounds are those of surface tissues, including a rupture, anabrasion, a surgical incision, a skin ulcer or a burn. Such skin woundsare treated by applying an emergency treatment to a wounded site andwaiting for the wounds to spontaneously heal via the biologicalrecovering power of their own. Such spontaneous healing requires a longtime until recovery and pain continues during the term. Therefore, it ispreferable that wound healing is actively promoted, by administering anagent for wound healing to wounded sites.

Because new epithelial tissues and connective tissues are formed throughcell migration and growth in the course of wound healing, apharmaceutical agent promoting or stimulating cell migration,differentiation and growth participating in wound healing is possibly anagent for wound healing. As such agent for wound healing, for example,lysozyme chloride and solcoseryl have been known.

However, the existent agents for wound healing do not have sufficientactions for promoting the wound healing so they are problematic in thatthey cannot completely heal wounds in a short period of time. It isconsidered that the cause is due to low contributions of these agentsfor wound healing to for example the re-surfacing of epidermis, collagensynthesis, the improvement of peripheral circulation, granulation, andangiogenesis, which are important elements in the course of woundhealing.

There is no report about the minimum unit for exhibiting the activity inIGF-I and there is no report about the peptide per se of the amino acidsequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1). Additionally,there is no report about the action of a joint administration of apeptide containing the amino acid sequence represented bySer-Ser-Ser-Arg (SEQ ID NO: 1) and a peptide containing the amino acidsequence represented by Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2) for cornealdisorders or the action thereof for skin diseases.

Generally, peptides consisting of numerous amino acids when administeredinto biological organisms are apt to be cut owing to metabolism and thelike. Additionally at a stage of their formulation for use aspharmaceutical agents, the peptides are apt to be decomposed. It isdesired that a peptide should have a chain as short as possible. Becausethe pharmacological activity thereof should be retained, however, it isan important subject in the development of pharmaceutical products tofind the minimum unit for the exhibition of the activity of a long-chainpeptide. IGF-I is a long-chain peptide consisting of amino acids as manyas 70. It is a very important subject for the preparation of a moreuseful pharmaceutical product to find the minimum unit for theexhibition of the activity of IGF-I. Still additionally, it is a veryinteresting subject to make studies about specific pharmacologicalactions, namely the action on corneal disorders and the action on skinwounds, using the minimum unit for the exhibition of the activity.

DISCLOSURE OF THE INVENTION

The present inventors found that the minimum unit for the exhibition ofthe activity of IGF-I was the amino acid sequence represented bySer-Ser-Ser-Arg (SEQ ID NO: 1) (referred to as “SSSR” hereinafter), bysynthesizing various partial peptides of IGF-I and carrying out apharmacological test about the extension of corneal epithelium,administering substance P or FGLM (SEQ ID NO: 2) in combination with thepartial peptides. The inventors also found that the joint administrationof a peptide containing the amino acid sequence represented by SSSR (SEQID NO: 1) and substance P or FGLM (SEQ ID NO: 2) could promote thecuring of corneal disorders and skin wound healing. Specifically, theinventors found that a composition containing (1) a peptide consistingof the amino acid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO:1), or an analog thereof or pharmaceutically acceptable salts thereofand (2) a peptide consisting of the amino acid sequence represented byPhe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2), or an analog thereof orpharmaceutically acceptable salts thereof was useful as a therapeuticagent for corneal disorders such as corneal ulcer, corneal erosion,keratitis or dry eye, where the cornea is at a damaged state because ofvarious factors and as a curing agent of skin wounds such as a rupture,an abrasion, a surgical incision, a skin ulcer or a burn and gangrenecaused by them. Herein, the therapeutic agent for corneal disorders andthe skin wounds healing promoting agent in accordance with the inventionmay be used in blend with ascorbic acid, ascorbic acid esters, ascorbicacid salts, pantothenic acid and pantothenic acid salts and the like,with their wound healing action having already been known.

IGF-I is composed of individual domains, A, B, C and D. The domains Aand B have a similar structure to those of insulin and IGF-II. Withattention focused on the domains C and D of IGF-I, thus, the inventorsexamined the action of extending corneal epithelium. Then, the inventorscarried out a corneal epithelium extension test, using the peptidecomposing the domain C or the peptide composing the domain D incombination with substance P. The inventors found that the peptidecomposing the domain C, namelyGly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 3) (referredto as “GYGSSSRRAPQT” hereinafter) had the activity. Even after two aminoacids were then removed from the two ends of GYGSSSRRAPQT (SEQ ID NO: 3)respectively, the activity still remained. Thus, the amino acids inGly-Ser-Ser-Ser-Arg-Arg-Ala-Pro (SEQ ID NO: 4) (referred to as“GSSSRRAP” hereinbelow) were sequentially substituted with alanine,using the alanine scanning approach, to synthesize alanine-substitutedamino acid sequences. In the presence of substance P or FGLM (SEQ ID NO:2) with the alanine-substituted amino acid sequences, then, a cornealepithelium extension test was carried out. Because all the peptidescontaining the amino acid sequence represented by SSSR (SEQ ID NO: 1)exhibited the activity, it was found that SSSR (SEQ ID NO: 1) was theessential, minimum partial peptide of IGF-I for the exhibition of theaction for corneal epithelium extension.

The inventors principally achieved the following four inventions.

A first invention relates to a novel peptide consisting of the aminoacid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1), or aderivative thereof or pharmaceutically acceptable salts thereof.

The feature of the first invention is based on the finding of the novelpeptide as the minimum unit for the activity exhibition of IGF-I, namelythe novel peptide consisting of the amino acid sequence represented bySer-Ser-Ser-Arg (SEQ ID NO: 1). Thus, the term peptide consisting of theamino acid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) or aderivative thereof (the peptide and a derivative thereof arecollectively referred hereinbelow to as “SSSR derivative”) means anynovel peptide containing the amino acid sequence represented bySer-Ser-Ser-Arg (SEQ ID NO: 1), with no specific limitation. Thederivative of the peptide means the peptide represented bySer-Ser-Ser-Arg (SEQ ID NO: 1) to which one or plural amino acids withno influence of the activity exhibition are preliminarily bound, thepeptide with the N terminal protected with protective groups widely usedfor peptides, such as acyl group, the peptide with the C terminalprotected with protective groups widely used for peptides, such as esterand amide. Additionally, the term derivative also includes the peptidewith the hydroxyl group in the Ser residue being protected with commonprotective groups or with the amino group in the Arg residue beingprotected with common protective groups. More specifically, the SSSR(SEQ ID NO: 1) derivative includes for example SSSR (SEQ ID NO: 1) andGSSSRRAP (SEQ ID NO: 4) and additionally includes for exampleSer-Ser-Ser-Arg-Arg (SEQ ID NO: 5) (abbreviated as “SSSRR” hereinbelow),Gly-Ser-Ser-Ser-Arg (SEQ ID NO: 6) (abbreviated as “GSSSR” hereinbelow),Gly-Ser-Ser-Ser-Arg-Arg (SEQ ID NO: 7) (abbreviated as “GSSSRR”hereinbelow), Ala-Ser-Ser-Ser-Arg-Arg-Ala-Pro (SEQ ID NO: 8)(abbreviated as “ASSSRRAP”), Gly-Ser-Ser-Ser-Arg-Ala-Ala-Pro (SEQ ID NO:9) (abbreviated as “GSSSRAAP” hereinbelow) andGly-Ser-Ser-Ser-Arg-Ala-Ala-Ala-Pro (SEQ ID NO: 10) (abbreviated as“GSSSRAAAP” hereinbelow). The amino acids composing these peptides arein L forms, D forms and DL forms, which are also encompassed within thescope of the invention. As specifically described in the sectionpharmacological test, all SSSR (SEQ ID NO: 1) derivatives containing theamino acid sequence represented by SSSR in the peptide chains when usedin combination with substance P or FGLM (SEQ ID NO: 2) can exhibit theeffect of extending corneal epithelium and the effect of promoting theskin wound healing.

The SSSR (SEQ ID NO: 1) derivatives of the invention can be prepared byknown methods using an automatic peptide synthesizer, and the detailsare described in the Examples.

A second invention relates to a pharmaceutical composition containingthe novel peptide consisting of the amino acid sequence represented bySer-Ser-Ser-Arg (SEQ ID NO: 1) or a derivative thereof orpharmaceutically acceptable salts thereof as an active ingredient andbeing blended with a pharmaceutically acceptable additive.

A third invention relates to an agent for treating a corneal disorder,the agent containing (1) a peptide consisting of the amino acid sequencerepresented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) or an analog thereof orpharmaceutically acceptable salts thereof and (2) a peptide consistingof the amino acid sequence represented by Phe-Gly-Leu-Met-NH₂ (SEQ IDNO: 2)or an analog thereof or pharmaceutically acceptable salts thereofas active ingredients.

A fourth invention relates to an agent for promoting skin wound healing,the agent containing the peptide (1) and the peptide (2) as activeingredients.

The feature of the third and fourth inventions is the finding that thejoint administration of the peptide where the minimum unit for theexhibition of the activity is represented by Ser-Ser-Ser-Arg (SEQ IDNO: 1) and the peptide where the minimum unit for the exhibition of theactivity is represented by Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2), canexhibit a excellent effect of extending corneal epithelium and also aexcellent effect of promoting skin wound healing.

In the third and fourth inventions, the term peptide consisting of theamino acid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) or ananalog thereof (the peptide and an analog thereof are collectivelyreferred to as “SSSR analog” hereinafter) means any peptide containingthe amino acid sequence represented by Ser-Ser-Ser-Arg (SEQ ID NO: 1),with no specific limitation. The analog of the peptide means the peptiderepresented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) to which one or pluralamino acids with no influence of the activity exhibition arepreliminarily bound, the peptide with the N terminal protected withprotective groups widely used for peptides, such as acyl group, thepeptide with the C terminal protected with protective groups widely usedfor peptides, such as ester and amide. Additionally, the term SSSR (SEQID NO: 1) analog also includes the peptide with the hydroxyl group inthe Ser residue being protected with common protective groups or withthe amino group in the Arg residue being protected with commonprotective groups. More specifically, the SSSR (SEQ ID NO: 1) analogincludes for example the SSSR (SEQ ID NO: 1) derivatives described aboveand GYGSSSRRAPQT (SEQ ID NO: 3). The amino acids composing the peptideof the SSSR (SEQ ID NO: 2) analog are in L forms, D forms and DL forms,which are all encompassed within the scope of the invention. Amorepreferable mode is a peptide composed of amino acids all in L forms.

Still additionally, the term peptide consisting of the amino acidsequence represented by Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2) or an analogthereof (the peptide and the analog thereof are collectively referred toas “FGLM analog” hereinafter) means any peptide containing the aminoacid sequence represented by Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2), with nospecific limitation. The analog of the peptide means the peptiderepresented by Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2) to which one or pluralamino acids with no influence of the activity exhibition arepreliminarily bound, and the peptide with the N terminal protected withprotective groups widely used for peptides, such as acyl group. Morespecifically, the FGLM (SEQ ID NO: 2) analog includes for examplesubstance P and FGLM (SEQ ID NO: 2) and additionally includes thefollowing polypeptides composed of four to 12 amino acids as disclosedin U.S. Pat. No. 3,862,114:

(SEQ ID NO: 11) Tyr-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- NH₂;(SEQ ID NO: 12) Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂; (SEQ ID NO: 13)Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂; (SEQ ID NO: 14)Gln-Phe-Phe-Gly-Leu-Met-NH₂; (SEQ ID NO: 15) Phe-Phe-Gly-Leu-Met-NH₂;(SEQ ID NO: 16) Tyr-Phe-Gly-Leu-Met-NH₂; and (SEQ ID NO: 17)Gly-Phe-Gly-Leu-Met-NH₂.

A preferable example thereof includes substance P and FGLM (SEQ ID NO:2). Amino acids composing them are in L forms, D forms and DL forms.They are all encompassed within the scope of the invention. A morepreferable mode is a peptide composed of amino acids all in L forms.

In accordance with the invention, pharmaceutically acceptable saltsthereof include for example hydrochloride salt, sulfate salt, phosphatesalt, lactate salt, maleate salt, fumarate salt, oxalate salt,methanesulfonate salt, and p-toluenesulfonate salt.

In accordance with the invention, the joint administration of the SSSR(SEQ ID NO: 1) analog and the FGLM (SEQ ID NO: 2) analog exhibitsactions of extending corneal epithelium and promoting the skin woundhealing. Any types of the SSSR (SEQ ID NO: 1) analog and the FGLM (SEQID NO: 2) analog exhibiting these actions are satisfactory with nospecific limitation. The joint administration of SSSR (SEQ ID NO: 1)asthe minimum unit of the activity exhibition of IGF-I and FGLM (SEQ IDNO: 2) as the minimum unit of the activity exhibition of substance P ispreferable for carrying out the invention.

The agent for treating corneal disorders and the agent for promoting theskin wound healing in accordance with the invention can be preparedusing common techniques. The SSSR (SEQ ID NO: 1) analog orpharmaceutically acceptable salts thereof and the FGLM (SEQ ID NO: 2)analog or pharmaceutically acceptable salts thereof are individuallyformulated into single formulations or formulated into blendformulations, which may be administered parenterally or orally.Parenteral administration thereof is more preferable.

Preferable dosage forms of the agent for treating corneal disordersinclude for example eye drops and eye ointments. These can be preparedusing common techniques. For example, the eye drops can be prepared,using isotonic agents such as sodium chloride, buffers such as sodiumphosphate, and preservatives such as benzalkonium chloride. The pH issatisfactory if it is within an ophthalmologically acceptable range.Preferred pH is within pH 4 to 8.

The dose of the agent for treating corneal disorders is appropriatelyselected, depending on the symptoms, age of patients, dosage form andthe like. For eye drops, the concentration of the SSSR (SEQ. ID NO: 1)analog or pharmaceutically acceptable salts thereof is 0.001 to 10 w/v%, preferably 0.01 to 1 w/v % for administration into eyes once orseveral times a day. The concentration of the FGLM (SEQ ID NO: 2) analogor pharmaceutically acceptable salts thereof is 0.00001 to 0.1 w/v %,preferably 0.0001 to 0.01 w/v % for administration into eyes once orseveral times a day.

It is needless to say that both the active ingredients are blendedtogether to prepare formulations such as eye drops.

Preferable forms of the formulation of the agent for promoting the skinwound healing include for example an ointment, a jelly, a cataplasm, apatch, a lotion, a cream, a spray, an aerosol, a plaster, a suspensionand an emulsion. Additionally, a liquid can be prepared by selecting anappropriate solvent. So as to prepare the agent for promoting the skinwound healing, the following agents can be added, depending on thedosage form: fillers, excipients, bases or vehicles, expanders, pHadjusters, solubilizers, suspending agents, buffers, stabilizers,preservatives, surfactants, anti-oxidants, dispersants, emulsifyingagents, dissolution agents and auxiliary agents for dissolution.

The carrier for the formulation includes for example white Vaseline,fluid paraffin, gelled hydrocarbon, cetyl alcohol, polyethylene glycol,gelatin, corn starch, sodium alginate, methyl cellulose, hydroxyethylcellulose, carboxymethyl cellulose, plastibase hydrophilic, gelatin,dextrin, cetyl alcohol, stearyl alcohol, polyethylene glycol, polyvinylalcohol, methoxyethylene-maleic anhydride copolymer, polyvinyl ether,and polymers and copolymers with a constitutional component of vinylpyrrolidone, sodium stearate, magnesium stearate, benzalkonium chloride,fats and oils such as olive oil, camellia oil, and soybean oil, lactoseand water.

The agent for promoting the skin wound healing in accordance with theinvention can be administered in various forms, depending on the woundsite and the wounded level. In case that the agent is to be used as anexternal preparation, the agent is preferably directly coated, sprayedor attached on a necessary site (lesion) such as skin.

The dose of the agent for promoting the skin wound healing in accordancewith the invention can be selected appropriately, in terms of thesymptoms, age of patients, dosage form and the like. The dose of theSSSR (SEQ ID NO: 1) analog or pharmaceutically acceptable salts thereofis generally 0.001 to 1000 mg, preferably 0.01 to 500 mg per day, in oneportion or in several portions. Additionally, the dose of the FGLM (SEQID NO: 2) analog or pharmaceutically acceptable salts thereof isgenerally 0.01 to 5000 mg, preferably 0.1 to 1000 mg per day, in oneportion or in several portions.

It is needless to say that both the active ingredients are blendedtogether to prepare formulations such as ointments.

Preparation examples, formulation examples and the results of apharmacological test are shown below. These are for better understandingof the invention but never limit the scope of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION Preparation Examples

Representative preparation examples of the SSSR analog for use in theinvention are shown below.

1. SSSR (SEQ ID NO: 1) Preparation

Using an automatic peptide synthesizer 430A (manufactured by AppliedBiosystems) and according to an existent software, a protective peptideresin was synthesized by the tertiary butyloxycarbonyl (BOC) method. Asa starting raw material, 4-(oxymethyl)phenylacetoamide methyl [Boc-Arg(Tos) PAM] resin carrier (0.5 mmol scale) was used. In this syntheticmethod, 30% trifluoroacetic acid (TFA)/dichloromethane and 70%TFA/dichloromethane were used for the removal of Boc group as a Na-aminoprotective group. For rinsing, N-methyl-2-pyrrolidone(NMP)/dichloromethane was used. N,N′-Dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBt) as condensing agents and the Boc-Ser(OBzl) derivative as an N-protected amino acid are used at 4 equivalentsper amino group respectively, while dimethylsulfoxide (DMSO)-NMP (8:2)was used as a reaction solvent. After completion of the condensation,the generation of defective peptides was prevented with aceticanhydride/N,N-diisopropylethylamine (DIEA) to completely block theremaining amino groups. The removal of the Boc group and thecondensation of Boc-Ser (OBzl) were repeated to construct the finalprotected peptide. The scissoring out of the peptide from the resultingprotected peptide resin and the elimination of all the protective groupswere carried out by a process with anhydrous hydrogen fluoride (HF) (HF:p-cresol=8:2 (v/v); −2 to −5° C.; 60 minutes). After the reaction, HFwas distilled away, and the peptide was extracted with aqueous 0.1%trifluoroacetic acid. A crude product was obtained as a freeze-driedpowder, for preparative separation and purification. The preparativeseparation and purification was done on a 0.5 to 2% gradient of anacetonitrile/water system (containing 0.1% TFA), using HPLC LC8A(manufactured by Shimadzu Corporation) (column: ODS 30×240 mmmanufactured by YMC) (80 minutes). After collecting highly purefractions of the resulting objective material and distillingacetonitrile away from the material, the resulting material wasfreeze-dried to obtain the TFA salt of the target compound (70 mg; yieldof 32%). Amino acid analysis (conditions for hydrolysis: 6N HCl, 110°C., 22 hours)

Ser(3) 2.74, Arg (1) 1.00

HPLC analysis [Column: YMC Pak ODS-A (4.6 mm I.D.×150 mm); Eluent: 1-60%CH₃CN/5 mM CF₃CF₂COOH (25 min); Temp.: 25° C.; Flow rate: 1.0 ml/min:Detector: 220 nm].

Purity (HPLC): 98.5%

Mass analysis (ESI-MS)

MH⁺=436.2 (Theor.=436.2, mono isotopic)

2. Preparation of SSSR (SEQ ID NO: 1) Analog

The same procedures for SSSR (SEQ ID NO: 1) were repeated to prepareGSSSR (SEQ ID NO: 6), SSSRR (SEQ ID NO: 5), GSSSRR (SEQ ID NO: 7),GSSSRRAP (SEQ ID NO: 4), ASSSRRAP (SEQ ID NO: 8), GSSSRAAP (SEQ ID NO:9) and GSSSRAAAP (SEQ ID NO: 10). The physical properties ofrepresentative peptides are shown below.

(1) GSSSR (SEQ ID NO: 6)Amino acid analysis (conditions for hydrolysis: 6N HCl, 110° C., 22hours)

Ser(3) 2.76, Gly (1) 1.00, Arg (1) 1.00

HPLC analysis [Column: YMC Pak ODS-A (4.6 mm I.D.×150 mm); Eluent: 1-60%CH₃CN/5 mM CF₃CF₂COOH (25 min); Temp.: 25° C.; Flow rate: 1.0 ml/min:Detector: 220 nm].

Purity (HPLC): 98.5%

Mass analysis (ESI-MS)

MW=492.3 (Theor.=492.5)

(2) SSSRR (SEQ ID NO: 5)Amino acid analysis (conditions for hydrolysis: 6N HCl, 110° C., 22hours)

Ser(3) 2.76, Arg (2) 2.00

HPLC analysis [Column: YMC Pak ODS-A (4.6 mm I.D.×150 mm); Eluent: 1-60%CH₃CN/0.1% CF₃COOH (25 min); Temp.: 25° C.; Flow rate: 1.0 ml/min:Detector: 220 nm].

Purity (HPLC): 99.7%

Mass analysis (ESI-MS)

MW=591.5 (Theor.=591.6)

(3) GSSSRR (SEQ ID NO: 7)Amino acid analysis (conditions for hydrolysis: 6N HCl, 110° C., 22hours)

Ser(3) 2.73, Gly(1) 0.98, Arg (2) 2.00

HPLC analysis [Column: YMC Pak ODS-A (4.6 mm I.D.×150 mm); Eluent: 1-60%CH₃CN/0.1% CF₃COOH (25 min); Temp.: 25° C.; Flow rate: 1.0 ml/min:Detector: 220 nm].

Purity (HPLC): 99.3%

Mass analysis (ESI-MS)

MW=648.5 (Theor.=648.7)

(4) GSSSRRAP (SEQ ID NO: 4)Amino acid analysis (conditions for hydrolysis: 6N HCl, 110° C., 22hours)

Ser(3) 2.68, Gly(1) 0.99, Ala(1) 1.01, Arg (2) 2.00

HPLC analysis [Column: YMC Pak ODS-A (4.6 mm I.D.×150 mm); Eluent: 1-60%CH₃CN/0.1% CF₃COOH (25 min); Temp.: 25° C.; Flow rate: 1.0 ml/min:Detector: 220 nm].

Purity (HPLC): 98.6%

Mass analysis (ESI-MS)

MW=816.7 (Theor.=816.9)

Formulation Examples

Representative formulation examples for use in accordance with theinvention are shown below.

1. Eye Drop

An eye drop of the following formulation was prepared by a wide method.

Formulation Example 1

SSSR   1 mg (SEQ ID NO: 1) Sodium chloride 900 mg Sodium hydroxidequantum sufficient Hydrochloric acid quantum sufficient Sterile purifiedquantum sufficient water In 100 ml

In the same manner as for the Formulation Example 1, eye dropscontaining SSSR (SEQ ID NO: 1) of 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 5mg, 10 mg, 50 mg and 100 mg in 100 ml can be prepared.

2. Formulation Example 2

GSSSR   1 mg (SEQ ID NO: 6) FGLM 100 mg (SEQ ID NO: 2) Sodium chloride900 mg Sodium hydroxide quantum sufficient Hydrochloric acid quantumsufficient Sterile purified quantum sufficient water In 100 ml

In the same manner as for the Formulation Example 2, eye dropscontaining FGLM (SEQ ID NO: 2)of 1 mg, 5 mg, 10 mg, 50 mg, 500 mg, and1000 mg in 100 ml can be prepared.

Formulation Example 3

SSSR   1 mg (SEQ ID NO: 1) FGLM 100 mg (SEQ ID NO: 2) Sodium chloride900 mg Sodium hydroxide quantum sufficient Hydrochloric acid quantumsufficient Sterile purified quantum sufficient water In 100 ml

In the same manner as for the Formulation Example 3, eye dropscontaining SSSR (SEQ ID NO: 1) of 0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 10mg, 50 mg and 100 mg and FGLM (SEQ ID NO: 2)of 1 mg, 5 mg, 10 mg, 50 mg,500 mg and 1000 mg in optional combinations can be prepared.

2. Ointment

Formulation Example 4 SSSR  10 mg (SEQ ID NO: 1) FGLM 100 mg (SEQ IDNO:2) Liquid paraffin  10 g White Vaseline quantum sufficient In 100 g

By appropriately modifying the amount of SSSR (SEQ ID NO: 1) to be addedand the amount of FGLM (SEQ ID NO: 2)to be added, various concentrationsof ointments can be prepared.

Formulation Example 5

GSSSR   1 mg (SEQ ID NO: 6) Substance P 100 mg Liquid paraffin  10 gWhite Vaseline quantum sufficient In 100 g

By appropriately modifying the amount of GSSSR (SEQ ID NO: 6) to beadded and the amount of substance P to be added in the same manner asfor the Formulation Example 5, various concentrations of ointments canbe prepared.

Formulation Example 6 SSSRR   5 mg (SEQ ID NO: 5) FGLM 100 mg (SEQ IDNO: 2) Liquid paraffin  10 g White Vaseline quantum sufficient In 100 g

By appropriately modifying the amount of SSSRR (SEQ ID NO: 5) to beadded and the amount of FGLM (SEQ ID NO: 2)to be added, variousconcentrations of ointments can be prepared.

Formulation Example 7

GSSSRR 50 mg (SEQ ID NO: 7) Substance P 10 mg Ascorbic acid  3 mg Liquidparaffin 10 g Plastibase hydrophilic quantum sufficient In 100 g

By appropriately modifying the amount of GSSSRR (SEQ ID NO: 7) to beadded and the amount of substance P to be added, various concentrationsof ointments can be prepared.

<Pharmacological Test>

(1) Action For Extending Corneal Epithelium (In Vitro)

Using the cornea of a male Japanese White rabbit and according to themethod of Nishida et al. (J. Cell Biol., 97, 1653-1657 (1983)), thelength of corneal epithelium extension of the corneal section in atissue culture system was used as a marker to examine the influence oncorneal epithelium extension.

Experimental Method

Corneal blocks cut off from rabbit corneal section (6 blocks per group)were cultured in culture media (TC-199) containing a test compound underconditions of 5% CO₂ at 37° C. for 24 hours. After culturing, thecorneal blocks were fixed in a mix solution of ethanol-glacial aceticacid (volume ratio: 95:5) and then embedded in paraffin to preparesections. After paraffin was removed from the sections, the resultingsections were stained with hematoxylin-eosin to examine the extendedlength of the corneal epithelial cell layer with a microscope. As acontrol, the blocks cultured in the same manner in the culture mediawithout any test compound were used.

Test Compounds

Representative examples of the peptides used in the experiment are shownin Table 1.

Results

The experimental results are shown in Table 1. Herein, the extensionratio in the table is the mean of six sections per group, as calculatedwhen the elongated length of the control group was defined as the basalline (100%).

TABLE 1 Extension Test drugs ratio (%) Control 100 SSSR 101 (SEQ IDNO: 1) (1 nM) GSSSR 101 (SEQ ID NO: 6) (1 nM) SSSRR 98 (SEQ ID NO: 5) (1nM) GSSSRR 94 (SEQ ID NO: 7) (1 nM) GSSSRRAP 97 (SEQ ID NO: 4) (1 nM)GSSSRAAAP 100 (SEQ ID NO: 10) (1 nM) GYGSSSRRAPQT 104 (SEQ ID NO: 3) (1nM) Substance P (20 μM) 94 FGLM 99 (SEQ ID NO: 2) (20 μM) SSSR 138 (SEQID NO: 1) (1 nM) + substance P (20 μM) GSSSR 135 (SEQ ID NO: 6) (1 nM) +substance P (20 μM) SSSRR 136 (SEQ ID NO: 5) Cl nM) + substance P (20μM) GSSSRR 142 (SEQ ID NO: 7) (1 nM) + substance P (20 μM) GSSSRRAP 140(SEQ ID NO: 4) (1 nM) + substance P (20 μM) ASSSRRAP 134 (SEQ ID NO: 8)(1 nM) + substance P (20 μM) GSSSRAAP 150 (SEQ ID NO: 9) (1 nM) +substance P (20 μM) GSSSRAAAP 139 (SEQ ID NO: 10) (1 nM) + substance P(20 μM) GYGSSSRRAPQT 134 (SEQ ID NO: 3) (1 nM) + substance P (20 μM)SSSR 145 (SEQ ID NO: 1) (1 nM) + FGLM (SEQ ID NO: 2) (20 μM)

As shown in Table 1, the SSSR (SEQ ID NO: 1) analogs alone, substance Palone and FGLM (SEQ ID NO: 2) alone were not observed to have anyinfluence on the extension of corneal epithelium; however, it wasobserved that the extension of corneal epithelium was significantlypromoted when the corneal epithelium was cultured in the culture mediumcontaining both the SSSR (SEQ ID NO: 1)analog and substance P (or FGLM(SEQ ID NO: 2)).

(2) Action On Healing Skin Wounds

The action of healing skin wounds can be tested by the following method.

A rat is anesthetized under inhalation of diethyl ether; then, thedorsal hair is razored with hair clippers and then removed with adepilatory cream. 24 hours later, five wound sites throughout all thelayers of epidermis and dermis are made at an equal interval on thedorsal skin, using a trephine of a 5 mm diameter for dermal biopsy.After hemostasis was confirmed, an SSSR (SEQ ID NO: 1)-containingointment, an FGLM (SEQ ID NO: 2)-containing ointment and anointmentcontaining SSSR (SEQ ID NO: 1) and FGLM (SEQ ID NO: 2) are individuallyapplied once daily. Before the application of the individual ointments,the dorsal wounds of the rat are photographed and measured of theirareas. The areas of the individual wounds after applied with the SSSR(SEQ ID NO: 1)-containing ointment, the FGLM (SEQ ID NO: 2)-containingointment and the ointment containing SSSR (SEQ ID NO: 1) and FGLM (SEQID NO: 2) are compared to each other, to examine the effect of healingskin wounds.

INDUSTRIAL APPLICABILITY

Based on the results of the pharmacological test, a joint administrationof the SSSR (SEQ ID NO: 1) analog containing the amino acid sequencerepresented by Ser-Ser-Ser-Arg (SEQ ID NO: 1) as the minimum unit forthe exhibition of the activity of IGF-I and the FGLM (SEQ ID NO: 2)analog containing the amino acid sequence represented byPhe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2) significantly promotes the extensionof corneal epithelium and the healing skin wounds. Thus, the SSSR (SEQID NO: 1) analog and the FGLM (SEQ ID NO: 2) analog when administered incombination synergistically act to exhibit effects as therapeutic agentsof corneal disorders such as corneal ulcer, corneal erosion, keratitisand dry eye or effects as healing agents of skin wounds such as arupture, an abrasion, a surgery incision, a skin ulcer and a burn anddiseases due to them, such as gangrene.

1. A method for treating a corneal disorder comprising administering to a patient in need thereof a composition containing pharmaceutically effective amounts of (i) a peptide of which the amino acid sequence is Ser-Ser-Ser-Arg (SEQ ID NO: 1), Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 3), Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro (SEQ ID NO: 4), Ser-Ser-Ser-Arg-Arg (SEQ ID NO: 5), Gly-Ser-Ser-Ser-Arg (SEQ ID NO: 6), Gly-Ser-Ser-Ser-Arg-Arg (SEQ ID NO: 7), Ala-Ser-Ser-Ser-Arg-Arg-Ala-Pro (SEQ ID NO: 8), Gly-Ser-Ser-Ser-Arg-Ala-Ala-Pro (SEQ ID NO: 9), Gly-Ser-Ser-Ser-Arg-Ala-Ala-Ala-Pro (SEQ ID NO: 10) or a pharmaceutically acceptable salt thereof and (ii) a peptide of which the amino acid sequence is —NH₂ Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2), substance P or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
 2. The method according to claim 1, wherein the corneal disorder is a corneal ulcer, a corneal erosion, keratitis or dry eye.
 3. The method according to claim 2, wherein the composition is administered in a dosage form which is an eye drop.
 4. A method for treating a corneal disorder comprising administering to a patient in need thereof a composition containing pharmaceutically effective amounts of (i) a peptide of which the amino acid sequence is Ser-Ser-Ser-Arg (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof and (ii) a peptide of which the amino acid sequence is Phe-Gly-Leu-Met-NH₂ (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
 5. The method according to claim 4, wherein the corneal disorder is a corneal ulcer, a corneal erosion, keratitis or dry eye.
 6. The method according to claim 4, wherein the composition is administered in a dosage form which is an eye drop. 